Statin derivatives

ABSTRACT

Statin nitroderivatives having improved pharmacological activity and enhanced tolerability are described. They can be employed for treating and/or preventing several diseases, in particular coronary syndromes, neurodegenerative disorders as well as for reducing cholesterol levels.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application which claims the benefit ofpending U.S. patent application Ser. No. 10/849,561, filed May 20, 2004.The disclosure of the prior application is hereby incorporated herein inits entirety by reference.

The present invention relates to new statin derivatives. Moreparticularly, the present invention relates to statin nitroderivatives,pharmaceutical compositions containing them and their use ascholesterol-reducing drugs, as drugs having immunosuppressiveproperties, antioxidant, antithrombotic and anti-inflammatory activity,effects on endothelial function, and for treating and/or preventingacute coronary syndromes, stroke, neurodegenerative disorders, such asAlzheimer's and Parkinson's disease as well as autoimmune diseases, suchas multiple sclerosis.

With statins a class of compounds is intended, comprising as maincomponents lovastatin, simvastatin, pravastatin, mevastatin,fluvastatin, atorvastatin, rosuvastatin and cerivastatin (rivastatin).They possess a side group that is structurally similar to HMG-CoA(3-hydroxy-3-methylglutaryl coenzyme A). Fluvastatin, atorvastatin,rosuvastatin and cerivastatin are entirely synthetic compoundscontaining a heptanoic acid side chain, the remainders being fungalmetabolites.

It is known that the various statins are inhibitors of HMG-CoAreductase, an enzyme which catalyses an early, rate-limiting step incholesterol biosynthesis, reduce triglyceride levels and are alsoindicated for raising HDL-C levels (P. O. Bonetti et al., European HeartJournal (2003) 24, 225-248).

However, it is also known that statins exhibit adverse effects, such asfor example hepatopathy, possible carcinogenic potential, muscularside-effects and, above all, myopathy.

It was now object of the present invention to provide new derivatives ofstatins able not only to eliminate or at least reduce the side effectsassociated with these compounds, but also having an improvedpharmacological activity. It has been so surprisingly found that statinnitroderivatives have a significantly improved overall profile ascompared to native statins both in term of wider pharmacologicalactivity and enhanced tolerability. In particular, it has beenrecognized that the statin nitroderivatives of the present inventionexhibit a strong anti-inflammatory, antithrombotic and antiplateletactivity and can be furthermore employed for reducing cholesterol andtriglycerides levels, for raising HDL-C levels and for treating orpreventing acute coronary syndromes, stroke, peripheral vasculardiseases such as peripheral ischemia and all disorders associated withendothelial dysfunctions such as vascular complications in diabeticpatients and atherosclerosis. They should also be employed for treatingneurodegenerative and autoimmune diseases, such as Alzheimer's andParkinson's disease as well as multiple sclerosis.

Object of the present invention are, therefore, statin nitroderivativesof general formula (I) and pharmaceutically acceptable salts orstereoisomers thereof

wherein R is the statin residue exemplified below and Y is a suitablelinker group, X being as defined below.

According to the present invention, the statin residue R in formula (I)is selected from the group consisting of pravastatin, fluvastatin,cerivastatin, rosuvastatin and atorvastatin.

In particular, in the general formula (I) R, X and Y have the followingmeanings:

-   X is —O—, —S—, —NH— or —NHR¹—, R¹ being straight or branched alkyl    with 1 to 10 carbon atoms, preferably CH₃;-   R is-   Y is a bivalent radical having the following meaning:    -   a)    -   1 straight or branched C₁-C₂₀ alkylene, preferably C₁-C₁₀, being        optionally substituted with one or more of the substituents        selected from the group consisting of: halogen atoms, hydroxy,        —ONO₂ or T₀, wherein T₀ is —OC(O)(C₁-C₁₀ alkyl)-ONO₂ or        —O(C₁-C₁₀ alkyl)-ONO₂;    -   2 cycloalkylene with 5 to 7 carbon atoms into cycloalkylene        ring, the ring being eventually substituted with side chains T,        wherein T is straight or branched alkyl with from 1 to 10 carbon        atoms, preferably CH₃;-   b)-   c)    wherein n is an integer from 0 to 20, and n¹ is an integer from 1 to    20;-   d)    wherein:-   n¹ is as defined above and n² is an integer from 0 to 2;-   X₁═—OCO— or —COO— and R² is H or CH₃;-   e)    wherein:-   n¹, n², R² and X₁ are as defined above;-   Y¹ is —CH₂—CH₂— or —CH═CH—(CH₂)_(n) ²—;-   f)    wherein:-   n¹ and R² are as defined above, R³ is H or COCH₃;-   with the proviso that when Y is selected from the bivalent radicals    mentioned under b)-f), the —ONO₂ group is bound to —(CH₂)_(n) ¹;-   g)    wherein X₂ is —O— or —S—, n³ is an integer from 1 to 6, preferably    from 1 to 4, R² is as defined above;-   h)    wherein:-   n⁴ is an integer from 0 to 10;-   n⁵ is an integer from 1 to 10;-   R⁴, R⁵, R⁶, R⁷ are the same or different, and are H or straight or    branched C₁-C₄-alkyl, preferably R⁴, R⁵, R⁶, R⁷ are H;-   wherein the —ONO₂ group is linked to    wherein n⁵ is as defined above;-   Y² is an heterocyclic saturated, unsaturated or aromatic 5 or 6    members ring, containing one or more heteroatoms selected from    nitrogen, oxygen, sulfur, and is selected from

The term “C₁-C₂₀ alkylene” as used herein refers to branched or straightchain C₁-C₂₀ hydrocarbon, preferably having from 1 to 10 carbon atomssuch as methylene, ethylene, propylene, isopropylene, n-butylene,pentylene, n-hexylene and the like.

The term “C₁-C₁₀ alkyl” as used herein refers to branched or straightchain alkyl groups comprising one to ten carbon atoms, including methyl,ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl,octyl and the like.

The term “cycloalkylene” as used herein refers to ring having from 5 to7 carbon atoms including, but not limited to, cyclopentylene,cyclohexylene optionally substituted with side chains such as straightor branched (C₁-C₁₀)-alkyl, preferably CH₃.

The term “heterocyclic” as used herein refers to saturated, unsaturatedor aromatic 5 or 6 members ring, containing one or more heteroatomsselected from nitrogen, oxygen, sulphur, such as for example pyridine,pyrazine, pyrimidine, pyrrolidine, morpholine, imidazole and the like.

Another aspect of the present invention provides the use of thecompounds of formula (I) in combination with at least a compound used totreat cardiovascular disease selected from the group consisting of: ACEinhibitors, angiotensin II receptor antagonists, beta-adrenergicblockers, calcium channel blockers, antithrombotics such as aspirin,nitrosated ACE inhibitors, nitrosated angiotensin II receptorantagonists, nitrosated beta-adrenergic blockers and nitrosated aspirin.

Suitable ACE inhibitors, angiotensin II receptor antagonists,beta-adrenergic blockers, calcium channel blockers, antithrombotics aredescribed in the literature such as The Merck Index (13^(th) edition).

Suitable nitrosated compounds are disclosed in WO 98/21193 and WO97/16405.

The administration of the compounds above reported can be carried outsimultaneously or successively.

The present invention also provides pharmaceutical kits comprising oneor more containers filled with one or more of the compounds and/orcompositions of the present invention and one or more of the compoundsused to treat cardiovascular diseases reported above.

As stated above, the invention includes also the pharmaceuticallyacceptable salts of the compounds of formula (I) and stereoisomersthereof.

The compounds according to the present invention, when they contain inthe molecule one salifiable nitrogen atom, can be transformed into thecorresponding salts by reaction in an organic solvent such asacetonitrile, tetrahydrofuran with the corresponding organic orinorganic acids.

Examples of organic acids are: oxalic, tartaric, maleic, succinic,citric acids. Examples of inorganic acids are: nitric, hydrochloric,sulphuric, phosphoric acids. Salts with nitric acid are preferred.

The compounds of the invention which have one or more asymmetric carbonatoms can exist as optically pure enantiomers, pure diastereomers,enantiomers mixtures, diastereomers mixtures, enantiomer racemicmixtures, racemates or racemate mixtures. Within the object of theinvention are also all the possible isomers, stereoisomers and theirmixtures of the compounds of formula (I).

Preferred compounds are those of formula (I) wherein:

-   X is —O— or —S—;-   R is a statin residue as defined above;-   Y is a bivalent radical having the following meaning:-   a)    -   3 straight or branched C₁-C₁₀ alkylene, being optionally        substituted with T₀, wherein T₀ is as above defined;-   b)-   c)    wherein n is an integer from 0 to 5, and n¹ is an integer from 1 to    5;-   d)    wherein:-   n¹ is as defined above and n² is an integer from 0 to 2;-   X₁═—OCO— or —COO— and R² is H or CH₃;-   e)    wherein:-   n¹, n², R² and X₁ are as defined above;-   Y¹ is —CH═CH—;-   f)    wherein:-   n¹ is as defined above, R² is H or CH₃, R³ is H or COCH₃;-   with the proviso that when Y is selected from the bivalent radicals    mentioned under b)-f), the —ONO₂ group is bound to —(CH₂)_(n) ¹;-   g)    wherein X₂ is —O— or —S—, n³ is an integer from 1 to 4, preferably    1, R² is a hydrogen atom or CH₃;-   h)    wherein:-   n⁴ is an integer from 0 to 3;-   n⁵ is an integer from 1 to 3;-   R⁴, R⁵ , R⁶, R⁷ are the same and are H;-   and wherein the —ONO₂ group is linked to-   Y² is a 6 member saturated, unsaturated or aromatic heterocyclic    ring, containing one or more atoms of nitrogen and selected for    example from

The following are preferred compounds according to the presentinvention:

-   fluvastatin 4-(nitrooxy)butyl ester,-   fluvastatin 4-(nitrooxymetyl)benzyl ester,-   fluvastatin 3-(nitrooxymethyl)benzyl ester,-   fluvastatin 2-(nitrooxymethyl)benzyl ester,-   fluvastatin 4-(nitrooxymethyl)phenyl ester,-   fluvastatin 3-(nitrooxymethyl)phenyl ester,-   fluvastatin 2-(nitrooxymethyl)phenyl ester,-   fluvastatin 2-[2′-(nitrooxy)ethyloxy]ethyl ester, corresponding to    formula:-   fluvastatin    2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester,    corresponding to formula:-   pravastatin 4-(nitrooxy)butyl ester,-   pravastatin 4-(nitrooxymetyl)benzyl ester,-   pravastatin 3-(nitrooxymethyl)benzyl ester,-   pravastatin 2-(nitrooxymethyl)benzyl ester,-   pravastatin 4-(nitrooxymethyl)phenyl ester,-   pravastatin 3-(nitrooxymethyl)phenyl ester,-   pravastatin 2-(nitrooxymethyl)phenyl ester,-   pravastatin 2-[2′-(nitrooxy)ethyloxy]ethyl ester, corresponding to    formula:-   pravastatin    2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester,    corresponding to formula:-   cerivastatin 4-(nitrooxy)butyl ester,-   cerivastatin 4-(nitrooxymetyl)benzyl ester,-   cerivastatin 3-(nitrooxymethyl)benzyl ester,-   cerivastatin 2-(nitrooxymethyl)benzyl ester,-   cerivastatin 4-(nitrooxymethyl)phenyl ester,-   cerivastatin 3-(nitrooxymethyl)phenyl ester,-   cerivastatin 2-(nitrooxymethyl)phenyl ester,-   cerivastatin 2-[2′-(nitrooxy)ethyloxy]ethyl ester, corresponding to    formula:-   cerivastatin    2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester,    corresponding to formula:-   atorvastatin 4-(nitrooxy)butyl ester,-   atorvastatin 4-(nitrooxymetyl)benzyl ester,-   atorvastatin 3-(nitrooxymethyl)benzyl ester,-   atorvastatin 2-(nitrooxymethyl)benzyl ester,-   atorvastatin 4-(nitrooxymethyl)phenyl ester,-   atorvastatin 3-(nitrooxymethyl)phenyl ester,-   atorvastatin 2-(nitrooxymethyl)phenyl ester,-   atorvastatin 2-[2′-(nitrooxy)ethyloxy]ethyl ester, corresponding to    formula:-   atorvastatin    2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester,    corresponding to formula:-   rosuvastatin 4-(nitrooxy)butyl ester,-   rosuvastatin 4-(nitrooxymethyl)benzyl ester,-   rosuvastatin 3-(nitrooxymethyl)benzyl ester,-   rosuvastatin 2-(nitrooxymethyl)benzyl ester,-   rosuvastatin 4-(nitrooxymethyl)phenyl ester,-   rosuvastatin 3-(nitrooxymethyl)phenyl ester,-   rosuvastatin 2-(nitrooxymethyl)phenyl ester,-   rosuvastatin 2-[2′-(nitrooxy)ethyloxy]ethyl ester, corresponding to    the formula:-   rosuvastatin    2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester,    corresponding to formula:

As mentioned above, object of the present invention are alsopharmaceutical compositions containing at least a compound of thepresent invention of formula (I) together with non toxic adiuvantsand/or carriers usually employed in the pharmaceutical field.

The daily dose of active ingredient that should be administered can be asingle dose or it can be an effective amount divided into severalsmaller doses that are to be administered throughout the day. Usually,total daily dose may be in amounts preferably from 50 to 500 mg. Thedosage regimen and administration frequency for treating the mentioneddiseases with the compound of the invention and/or with thepharmaceutical compositions of the present invention will be selected inaccordance with a variety of factors, including for example age, bodyweight, sex and medical condition of the patient as well as severity ofthe disease, route of administration, pharmacological considerations andeventual concomitant therapy with other drugs. In some instances, dosagelevels below or above the aforesaid range and/or more frequent may beadequate, and this logically will be within the judgment of thephysician and will depend on the disease state.

The compounds of the invention may be administered orally, parenterally,rectally or topically, by inhalation or aerosol, in formulationseventually containing conventional non-toxic pharmaceutically acceptablecarriers, adjuvants and vehicles as desired. Topical administration mayalso involve the use of transdermal administration such as transdermalpatches or iontophoresis devices. The term “parenteral” as used herein,includes subcutaneous injections, intravenous, intramuscular,intrasternal injection or infusion techniques.

Injectable preparations, for example sterile injectable aqueous oroleaginous suspensions may be formulated according to known art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent.Among the acceptable vehicles and solvents are water, Ringer's solutionand isotonic sodium chloride. In addition, sterile, fixed oils areconventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed including synthetic mono ordiglycerides, in addition fatty acids such as oleic acid find use in thepreparation of injectables.

Suppositories for rectal administration of the drug can be prepared bymixing the active ingredient with a suitable non-irritating excipient,such as cocoa butter and polyethylene glycols.

Solid dosage forms for oral administration may include capsules,tablets, pills, powders, granules and gels. In such solid dosage forms,the active compound may be admixed with at least one inert diluent suchas sucrose, lactose or starch. Such dosage forms may also comprise, asin normal practice, additional substances other than inert diluents,e.g. lubricating agents such as magnesium stearate. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. Tablets and pills can additionally be prepared withenteric coatings.

Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting agents,emulsifying and suspending agents, and sweetening, flavouring and thelike.

As previously reported, the new statin nitroderivatives beyond loweringlipid, possess enhanced anti-inflammatory, antiplatelet andantithrombotic effects as compared to native statins. Moreover, they canbe effective also in the other pathologies such as acute coronarysyndromes, stroke, peripheral vascular diseases such as peripheralischemia, all disorders associated with endothelial dysfunctions such asvascular complications in diabetic patients and atherosclerosis,neurodegenerative diseases such as Alzheimer's disease (AD) andParkinson's disease (PD), autoimmune diseases such as multiplesclerosis.

Study on Vascular Tone

The ability of statin nitroderivatives to induce vasorelaxation incomparison to native statins, was tested in vitro in isolated rabbitthoracic aorta preparations (Wanstall J. C. et al., Br. J. Pharmacol.,134:463-472, 2001). Male New Zealand rabbits were anaesthetized withthiopental-Na (50 mg/kg, iv), sacrificed by exsanguinations and then thethorax was opened and the aorta dissected. Single ring preparations (4mm in length) of thoracic aorta were set up in physiological saltsolution (PSS) at 37° C. in small organ chambers (5 ml). The compositionof PSS was (mM): NaCl 130, NaHCO₃ 14.9, KH₂PO₄ 1.2, MgSO₄ 1.2, HEPES 10,CaCl₂, ascorbic acid 170 and glucose 1.1 (95% O₂/5% CO₂; pH 7.4). Eachring was mounted under 2 g passive tension in 5 ml organ bath. Isometrictension was recorded with a Grass transducer (Grass FT03) attached to aBIOPAC MP150 System. Preparations were allowed to equilibrate for 1 h,then contracted submaximally with noradrenaline (NA, 1 μM) and, when thecontraction was stable, acetylcholine (ACh, 10 μM) was added. A relaxantresponse to ACh indicated the presence of a functional endothelium. Whena stable precontraction was reached, a cumulative concentration-responsecurve to either of the vasorelaxant agents was obtained in the presenceof a functional endothelium. Time intervals between differentconcentrations were based on the time needed to reach a full response.Moreover, the effect of the soluble guanylyl cyclase inhibitor ODQ(1-H-(1,2,4)-oxadiazol(4,3-a)quinoxalin-1-one) on the dilator responseselicited by the compounds was examined preincubating the aortic ringswith ODQ (10 μM) for 20 min. Responses to vasorelaxing agents weremeasured from the plateau of NA contraction. The IC₅₀ (concentrationgiving 50% reversal of NA contraction) was interpolated from the plotsof relaxant-response vs log molar concentration of tested compound.

During the experimental period, the plateau obtained with NA was stablewithout significant spontaneous loss of contraction in the aortic rings.Under these experimental conditions, native statins did not producerelaxation up to 30 μM, the curve being not different from that built upin presence of vehicle alone.

As shown in the following Table 1, the nitroderivatives of the inventionwere able to induce relaxation in a concentration-dependent manner.Furthermore, in experiments performed in presence of ODQ (10 μM), thevasorelaxant responses to all the tested drugs were inhibited. TABLE 1Compound IC₅₀ (μM) ± sem Pravastatin >30 Compound of EX. 1 2.4 ± 0.6Fluvastatin >30 Compound of EX. 3 13.4 ± 3.5  Compound of EX. 4 8.7 ±3.3IC₅₀ is the concentration which inhibits 50% of the response.Effects of Nitrostatin Derivatives on Inflammatory Pathways in Vitro

The experiments were performed using RAW264.7 monocyte macrophage cellline. Cells were stimulated in the presence of LPS (1 μg/ml) for 8 h(pravastatin and derivative) or 16 h (fluvastatin, atorvastatin andderivatives). At the end of the incubation, the cells were harvested inlysis buffer and the protein content was measured. Western Blot ofinducible iNOS and COX2 proteins (representatives of an ongoinginflammatory process) was performed. The obtained results reported inTable 2 are expressed as % reduction of optical density of eachtreatment vs. LPS-treated samples. Negative results indicate an increasevs. LPS alone. TABLE 2 iNOS (% COX2 (% Concentration reduction reductionCompound (μM) vs LPS) vs LPS) Fluvastatin 10 −14.8 ± 5.9  −2.8 ± 3.7Compound of EX. 3 10 44.8 ± 11.9 53.9 ± 6.1 Compound of EX. 4 10 22.4 ±9.6  19.4 ± 6.5 Pravastatin 100 4.7 ± 3.6 26.9 ± 7.9 Compound of EX. 1100 55.2 ± 11.8 74.9 ± 4.4 Atorvastatin 10 20 ± 14 — Compound of EX. 710 45 ± 9  —Nitroderivatives of fluvastatin, pravastatin and atorvastatin exert, inPAW264.7 cell line, a significant inhibitory action on the expression ofproteins, such as COX2 and iNOS, that are recognised to be relevant inthe inflammatory process. Conversely, these effects are not detectableor weak for the parent compounds.Study of the Activity of Pravastatin Nitroderivative on PeripheralVascular Disease

Angiogenesis is a primary response to local tissue hypoxia and is likelyinvolved in the restoration of blood flow in ischemic diseases such asacute coronary syndromes and occlusive peripheral vascular disease.

The ability of pravastatin nitroderivative (compound of EX.1) to inhibitthe angiogenic process was evaluated using an in vivo model of murinehindlimb ischemia as previously described (Emanueli et al., Circulation103,125-132, 2001). Briefly, 2 month-old CD1 male mice (Charles River,Italy) mice were randomly allocated into 3 groups (n=20 each) feed ratchow containing: 1) pravastatin (40 mg/kg), 2) pravastatinnitroderivative (48 mg/kg), 3) vehicle (regular chow). After 3 days,mice were anaesthetized and left hindlimb ischemia was induced byelectro-coagulation of the upper part of femoral artery. Treatment wascontinued for the following 2 weeks after surgery. Systolic bloodpressure was measured by tail cuff technique at 7 and 14 days. Theclinical outcome at 14 days was determined by counting the number ofnecrotic or lost nails in the ischemic foot as well as the number ofauto-amputation events. At 14 days from ischemia induction, hindlimbs ofanaesthetized mice were perfusion-fixed, both adductors were coded andhistologically processed. Capillary and arteriolar densities(n_(cap)/mm² and n_(art)/mm²) were determined. TABLE 3 N capillaries/mm²N capillaries/mm² in ischemic in contralateral Compound adductor muscleadductor muscle Vehicle  852 ± 32^(§§) 685 ± 38 Pravastatin  823 ± 42736 ± 40 Compound of EX. 1 1116 ± 53^(§§)**⁺⁺ 660 ± 40**P < 0.01 vs. vehicle;⁺⁺P < 0.01 vs. pravastatin;^(§§)P < 0.01 vs. respective contralateral groupAs shown in Table 3, differently from the parent compound, the compoundof the invention was able to potentiate the native reparativecapillarization response to ischemia. The results of the present studyindicate that the nitroderivative promotes a significant reparativeneovascularization and ameliorates clinical outcome in mice withexperimentally-induced hindlimb ischemia.Effect of Pravastatin Nitroderivative on Leukocytes Adhesion in ApoEKnockout Mice

The interaction between leukocytes and the vascular endothelium is acrucial inflammatory step in the atherogenic process. The ability ofpravastatin nitroderivative (compound of EX.1) to inhibit leukocyteadhesion was investigated ex vivo in atherosclerotic (ApoE knockout)mice.

Three groups of ApoE knockout mice were dosed with pravastatin (40 mg/kgpo), pravastatin nitroderivative (equimolar dose) or vehicle daily for 5days and euthanised by CO₂ euthanised by CO₂ asphyxiation 1 h after thelast dose. Leukocytes were isolated from spleen, centrifuged, RBCs lysedwith water. Arterial segments prepared—aortic & thoracic regions openedlongitudinally and pinned out luminal side up. Radiolabelled leukocytes(⁵¹Cr-leukocytes) are incubated with thrombin-stimulated (10 UmL−1 for10 min.) segments for 30 min. Segments are washed with medium andadhesion measured by gamma counter. TABLE 4 ApoE K/O mice Leukocyteadhesion (%) Compound (thoracic aorta) Vehicle 23.4 ± 5 Pravastatin 23.0± 6 Compound of EX. 1  9.3 ± 3*#*P < 0.05 vs vehicle;#P < 0.05 vs pravastatinAs shown in Table 4, differently from the parent compound, the compoundof the invention was able to reduce the thrombin-induced adhesiveinteraction between isolated leukocytes and the blood vessel wall inboth strains of mice.

These data demonstrate that the nitroderivative exertsanti-atherogenic/anti-inflammatory effects.

Study of Antithrombotic Activity of Pravastatin Nitroderivative in Vivo

The ability of pravastatin nitroderivative to inhibit thrombus formationwas evaluated in male Charles Rivers CD-1 mice (20-25 g) givenpravastatin (10 or 20 mg/kg ip), compound of EX.1 (equimolar doses) orvehicle. 1-3 hrs later the animals were injected into a tail vein withU46619 (0.2 mg/kg), a stable TxA2 analog. As previously described (Momiet al., Eur. J. Pharmacol. 397:177-185, 2000), this dose of the agonistcaused 80 to 90% mortality within 3 min. in the control group. In eachexperimental session at least six animals per treatment group weretested; control groups were run at the beginning and at the end of everyexperimental session. Protection against agonist-induced thromboembolismwas expressed as (1-T_(drug)/T_(saline))×100, were T_(drug) is themortality rate in drug-treated mice, and T_(saline) is the number of nonsurviving control animals which received thrombin only.

The results were expressed as percentage of protection fromU46619-induced pulmonary thromboembolism as compared to the controlgroup. TABLE 5 Antithrombotic Antithrombotic activity (%) activity (%)Compound 1-h before challenge 3-h before challenge Vehicle  0  0Pravastatin 10 mg/kg ip 15 10 20 mg/kg ip 25 20 Compound of EX. 1 12mg/kg ip  30*  40* 24 mg/kg ip  40*+  55*+*P < 0.05 vs control;+P < 0.05 vs pravastatinAs shown in Table 5, differently from the parent compound, the compoundof the invention was able to inhibit thrombosis induced by U46619.Study of Antiplatelet Activity of Pravastatin Nitroderivative in Vitro

Platelets are prominent components of the thrombi. The ability ofpravastatin nitroderivative (compound of EX.1) to inhibit plateletaggregation was evaluated in vitro in human platelets. Plateletaggregation was measured in 0.25 ml platelet reached plasma (PRP) or gelfiltered platelets (GFP) samples as previously described (Vezza R, etal., Blood 82: 2704-2713, 1993).

Compounds were incubated at 37° C. for 2 min before adding U46619, anaggregating agent, TxA₂ analog. Aggregation was followed for 5 min andthe maximal amplitude (cm) was measured. DMSO (0.05%) was used asvehicle. Compounds were tested at concentrations ranging from 50 to 200μM. TABLE 6 Platelet Platelet aggregation (GFP) aggregation (PRP)Compound IC₅₀ μM IC₅₀ μM Pravastatin >200 >200 Compound of EX. 1 101 ±10 74 ± 7As shown in Table 6, differently from the parent compound, thenitroderivative of the invention was able to inhibit plateletaggregation induced by U46619.Effect of Pravastatin Nitroderivative on Inhibition of Tissue FactorExpression in Vitro

Tissue factor (TF) is a major regulator of homeostasis and thrombosis.The ability of pravastatin nitroderivative (compound of EX.1) to inhibitTF expression was evaluated in isolated human blood mononuclear cell(MNC) preparations. MNC were obtained from fresh blood by densitygradient (Ficoll-Hypaque) centrifugation and resuspended in serum-freeRPMI medium at the concentration of 2-3×10⁶/ml. Pravastatinnitroderivative (3-50 μM) or pravastatin (50 μM) was added to cellsuspensions prior to exposure to LPS (1 μg/ml). After incubation for 3 hat 37° C., TF expression in MNC was assessed by 1) functional assay(Semeraro et al., 1983. Immunology; 50: 529-35) on intact cells; 2)immunological assay (Imubind, Instrumentation Laboratory, Milan) onTriton X-100 cell extracts; 3) RT-PCR (Rossiello et al. Thromb Haemost2000; 84:453-59). TABLE 7 TF in LPS-stimulated MNC Activity Antigen(AU/10⁶ cells)^(§) (ng/mg protein) Vehicle (DMSO 0.1%) 178 ± 36.2 5.0 ±1.1 Pravastatin (50 μM) 189 ± 37.6 4.4 ± 1.3 Compound of EX. 1 (50 μM) 16 ± 4.9**  0.65 ± 0.11**Data are the mean ± SEM of 4-5 experiments.^(§)Arbitrary units calculated by reference to a standard curveconstructed with human relipidated tissue factor**P < 0.01 vs pravastatin and vehicleAs shown in Table 7, differently from the parent compound, the compoundof the invention markedly inhibited LPS-induced TF expression (activityand antigen).

The results of the present study indicate that the nitroderivativeexerts antithrombotic activity.

The following examples are to further illustrate the invention withoutlimiting it.

General Procedure

The compound of general formula (I), wherein X═O, can be obtained byreacting a compound of formula (II)

wherein M is hydrogen or an alkali metal or alkaline earth metal, suchas sodium or calcium, with a compound of formula (III)A-Y—B   (III)wherein A is a leaving group such as chlorine, bromine, iodine, a tosylor mesyl group, B is selected from chlorine, bromine, iodine or anitrooxy group and Y is as defined above in formula (I), in an inertorganic solvent such as N,N′-dimethylformamide, tetrahydrofurane,benzene, toluene, at a temperature ranging from room temperature to 50°C. The reaction is terminated within 30 min to 24 h.

The compounds of formula (II) are known compounds available on themarket or can be prepared according to methods well known in theliterature. The compounds of formula (III), wherein A is as abovedefined and B is Cl, Br, I, are commercially available or can besynthesized according to methods well known in the literature.

The compounds of formula (III), wherein A is as above defined and B isONO₂, are synthesized by conversion of a compound of formula (III)wherein B is Cl, Br, I into the corresponding nitro derivative byreaction with a nitrate source such as silver nitrate, lithium nitrate,sodium nitrate, potassium nitrate, magnesium nitrate, calcium nitrate,iron nitrate, zinc nitrate or tetraalkylammonium nitrate (wherein alkylis C₁-C₁₀ alkyl) in a suitable organic solvent such as acetonitrile,tetrahydrofurane, methyl ethyl ketone, ethyl acetate, DMF, the reactionis carried out, in the dark, at a temperature from room temperature tothe boiling temperature of the solvent. The reaction is terminatedwithin 30 min to 3 days. Preferred nitrate source is silver nitrate.

The compound of formula (I), in which X═O, S, NH or NR¹, R¹ being asabove defined, can be obtained by reacting a compound of formula (II)wherein M is hydrogen, in presence of a dehydrating agent, with acompound of formula (IV)D-Y—B   (IV)wherein Y e B are as defined above and D is selected from NH₂, NHR¹, OHor SH, R¹ being as above defined. Preferred dehydrating agents areN,N′-carbonyldiimidazole, used in presence of a catalytic amount ofsodium ethylate, DCC EDAC. The reaction is performed in an inert organicsolvent such as N,N′-dimethylformamide, tetrahydrofurane, benzene,toluene, a polyhalogenated aliphatic hydrocarbon, at a temperatureranging from −50° C. to 50° C. for between 30 min and 24 h.

The compounds of formula (IV), wherein D is as above defined and B isCl, Br, I, are commercially available or synthesized according tomethods well known in the literature.

For example they can be obtained from the corresponding hydroxyderivative of formula (V) D-Y—OH, by reaction with thionyl or oxalylchloride, halides of P^(III) or P^(V), mesyl chloride, tosyl chloride insolvents inert such as toluene, chloroform, DMF, etc.

The compounds of formula (IV), wherein D is as above defined and B isONO₂, can be obtained by reaction of the compounds of formula (IV)wherein B is Cl, Br, I, with a nitrate source as above described.Alternatively the compounds of formula (IV) wherein B is ONO₂, can beobtained by the corresponding hydroxy compounds of formula (V) byreaction with nitric acid and acetic anhydride in a temperature rangefrom −50° C. to 0° C. according to methods well known in the literature.

EXAMPLE 1 Synthesis of[1S-[1α(βS*,δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-(nitrooxy)butyl ester (pravastatin 4-(nitrooxy)butyl ester)

a)[1S-[1α(βS*,δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-Hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-bromobutyl ester

To a solution of pravastatin sodium (9.25 g, 21 mmol) inN,N-dimethylformamide (70 ml) a mixture of 1,4-dibromobutane (3.68 ml,31 mmol) in N,N-dimethylformamide (30 ml) was added dropwise. Thereaction mixture was stirred at room temperature for 24 h. The solutionwas then treated with water and diethyl ether, the organic layers weredried with sodium sulfate and concentrated under reduced pressure. Theresidue was purified by flash chromatography, with n-hexane/ethylacetate 3/7 as eluent. The title compound was obtained as a white powder(7.8 g).

b)[1S-[1α(βS*δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-Hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-(nitrooxy)-butyl ester

A solution of the compound obtained as described in a) above (7.7 g, 14mmol) and silver nitrate (3.5 g, 21 mmol) in acetonitrile (90 ml) wasstirred at 40° C., in the dark, for 48 h. The precipitate thus formed(silver salt) was filtered off and the solvent was evaporated undervacuum. The residue was then purified by flash chromatography, withn-hexane/ethyl acetate 3/7 as eluent. The title compound was obtained asa white powder (3.7 g) melting at 63° C.

¹H-NMR: δ (CDCl₃) 6.00(1H,m); 5.88(1H,m); 5.53(1H,s); 5.40(1H,s);4.51(2H,t); 4.40(1H,m); 4.26(1H,m); 4.15(2H,t); 3.78(1H,m);2.60-2.23(6H,m); 1.8-1.20(14H,m); 1.10(3H,d); 0.87(6H,t).

EXAMPLE 2 Synthesis of[1S-[1α(βS*,δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-(nitrooxymethyl)benzyl ester (pravastatin4-(nitro-oxymethyl)benzyl ester)

a)1S-[1α(βS*δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-Hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-(chloro-methyl) benzyl ester

To a solution of α,α′-dichloro-p-xylene (5.5 g, 31 mmol) inN,N-dimethylformamide (70 ml) pravastatin sodium (7 g, 15 mmol) wasadded portionwise. The reaction mixture was stirred at room temperaturefor 24 h. The solution thus obtained was then treated with water andethyl acetate, the organic layers were anidrified with sodium sulfateand concentrated under reduced pressure. The residue was purified bysilica gel chromatography, with n-hexane/ethyl acetate 1/1 as eluent.The title compound was obtained as a white powder (5.5 g) and wasemployed as such in the following step without further purification.

b)[1S-[1α(βS*,δS*),2α,6α,8β-(R*),8aα]]-1,2,6,7,8,8a-Hexahydro-β,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthaleneheptanoicacid 4-(nitrooxymethyl) benzyl ester

A solution of the compound obtained as described in a) above (5.5 g, 7.7mmol) and silver nitrate (2.5 g, 15 mmol) in acetonitrile (60 ml) wasstirred at 60° C., in the dark, for 48 h. The precipitate thus formed(silver salt) was filtered off and the solvent was evaporated undervacuum. The residue was then purified by flash chromatography, withn-hexane/ethyl acetate 1/1 as eluent. The title compound (1.6 g) wasobtained as white powder melting at 80-82° C.

¹H-NMR δ(CDCl₃):7.39(4H,m); 5.98(1H,m); 5.86(1H,m); 5.54(1H,s);5.42(3H,s); 5.16(2H,s); 4.40(1H,m); 4.27(1H;m); 3.80(1H,m); 2.52(3H,m);2.45-2.25(3H,m); 1.74-1.10(10H,m); 1.10(3H,d); 0.87(6H,t).

EXAMPLE 3 Synthesis of[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-(nitrooxy)butyl ester (fluvastatin 4-(nitrooxy)butyl ester)

a)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-bromobutyl ester

To a solution of 1,4-dibromobutane (4.1 ml, 34 mmol) inN,N-dimethylformamide (60 ml) a mixture of fluvastatin sodium (5 g, 11mmol) in N,N-dimethylformamide (40 ml) was added dropwise. The reactionmixture was stirred at room temperature for 24 h. The resulting solutionwas then treated with water and diethyl ether, the organic layers wereanidrified with sodium sulfate and concentrated under reduced pressure.The residue was purified by flash chromatography, withn-hexane/iso-propanol 8.5/1.5 as eluent. The title compound was obtainedas yellow oil (4.7 g) and was employed in the following step withoutfurther purification.

b)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-(nitrooxy)butyl ester

A solution of the compound obtained as described in a) above (4.7 g, 8.5mmol) and silver nitrate (2.9 g, 17 mmol) in acetonitrile (60 ml) wasstirred at 60° C., in the dark, for 48 h. The precipitate thus formed(silver salt) was filtered off and the solvent was evaporated undervacuum. The residue was then purified by flash chromatography, withn-hexane/ethyl acetate 1/1 as eluent. The desired product (0.5 g) wasobtained as white powder melting at 112° C.

¹H-NMR δ (DMSO): 7.69(1H,d); 7.43(3H,m); 7.27(2H,t); 7.25(1H,t);7.03(1H,t); 6.65(1H,d); 5.75(1H,dd); 4.98(1H,d); 4.90(1H,m); 4.78(1H,d);4.50(2H,t); 4.25(1H,m); 4.09(2H,t); 3.9(1H,m); 2.4(2H,m); 1.67(4H,m);1.61(6H,d); 1.42(2H,m).

EXAMPLE 4 Synthesis of[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-(nitrooxymethyl)benzyl ester (fluvastatin4-(nitrooxymethyl)benzyl ester)

a)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-(chloromethyl)benzyl ester

To a solution of α,α′-dichloro-p-xylene (10 g, 57 mmol) inN,N-dimethylformamide (60 ml) a mixture of fluvastatin sodium (10 g, 23mmol) in N,N-dimethylformamide (80 ml) was added dropwise. The reactionmixture was stirred in the dark at room temperature for 12 h. Thesolution was then treated with water and ethyl acetate, the organiclayers were anidrified with sodium sulfate and concentrated underreduced pressure. The residue was purified by flash chromatography, withn-hexane/ethyl acetate 1/1 as eluent. The title compound (9.4 g) wasobtained as white powder by crystallization from n-hexane.

b)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 4-(nitrooxymethyl)benzyl ester

A solution of the compound described in a) above (9.4 g, 17 mmol) andsilver nitrate (10.6 g, 63 mmol) in acetonitrile (180 ml) was stirred at50° C., in the dark, for 24 h. The precipitate thus obtained (silversalt) was filtered off and the solvent was evaporated under vacuum. Theresidue was then purified by flash chromatography, with n-hexane/ethylacetate 4/6 as eluent to give the desired product (4 g) as a whitepowder melting at 103-104° C.

1H-NMR δ (DMSO): 7.65(1H,d); 7.42(7H,m); 7.22(2H,t); 7.15(1H,t);7.03(1H,t); 6.64(1H,d); 5.75(1H,dd); 5.51(2H,s); 5.12(2H,s); 4.98(1H,d);4.88(1H,t); 4.84(1H,d); 4.27(1H,t); 3.94(1H,t); 2.4-2.6(2H,m);1.4-1.7(2H,M); 1.57(6H,d).

EXAMPLE 5 Synthesis of[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 3-(nitrooxymethyl)benzyl ester (fluvastatin3-(nitrooxymethyl)benzyl ester)

a)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 3-(chloromethyl)benzyl ester

To a solution of α,α′-dichloro-m-xylene (0.95 g, 5.4 mmol) inN,N-dimethylformamide (25 ml) a mixture of fluvastatin sodium (0.79 g,1.8 mmol) in N,N-dimethylformamide (25 ml) was added dropwise. Thereaction mixture was stirred in the dark at room temperature for 24 h.The solution thus formed was then treated with water and ethyl acetate,the organic layers were anidrified with sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gelchromatography, with n-hexane/ethyl acetate 1/1 as eluent, to give 0.41g of the title compound.

b)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 3-(nitrooxymethyl)benzyl ester

A solution of the compound described above (0.4 g, 0.7 mmol) and silvernitrate (0.36 g, 2.1 mmol) in acetonitrile (30 ml) was stirred at 45°C., in the dark, for 24 h. The precipitate thus obtained (silver salt)was filtered off and the solvent was evaporated under vacuum. Theresidue was purified by flash chromatography, with methylenechloride/iso-propanol 9.5/0.5 as eluent to give the desired product (0.2g).

¹H-NMR: δ (DMSO): 7.67(1H,d); 7.47-7.40(7H,m); 7.25-7.10(3H,m);7.05(1H,t); 6.62(1H,d); 5.72(1H,dd); 5.49(2H,s); 5.13(2H,s);5.00-4.84(3H,m); 4.27(1H,m); 3.95(1H,m); 2.60-2.35(2H,m); 1.58(6H,d);1.70-1.45(2H,m).

EXAMPLE 6 Synthesis of[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methylethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 2-(nitrooxymethyl)benzyl ester (fluvastatin2-(nitrooxymethyl)benzyl ester)

a)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl)-3,5-dihydroxy-6-heptanoicacid 2-(chloromethyl)benzyl ester

To a solution of α,α′-dichloro-o-xylene (1.5 g, 3.4 mmol) inN,N-dimethylformamide (15 ml) a mixture of fluvastatin sodium (1.5 g,8.6 mmol) in N,N-dimethylformamide (15 ml) was added dropwise. Thereaction mixture was stirred in the dark at room temperature for 24 h.The solution thus obtained was treated with water and ethyl acetate, theorganic layers were anidrified with sodium sulfate and concentratedunder reduced pressure. The residue was purified by silica gelchromatography, with n-hexane/ethyl acetate 6/4 as eluent to give 0.97 gof the title compound.

b)[R*,S*-(E)]-7-[3-(4-fluorophenyl)-1-(1-methyl-ethyl)-1H-indol-2-yl]-3,5-dihydroxy-6-heptanoicacid 2-(nitrooxymethyl)benzyl ester

A solution of the compound obtained as described above (0.97 g, 1.7mmol) and silver nitrate (1.3 g, 7.6 mmol) in acetonitrile (40 ml) wasstirred at 40° C., in the dark, for 24 h. The precipitate thus formed(silver salt) was filtered off and the solvent was evaporated undervacuum. The residue was then purified by flash chromatography, withn-hexane/iso-propanol 8.5/1.5 as eluent to give the desired product(0.42 g) as yellow powder.

¹H-NMR δ (DMSO): 6.52 (1H,m); 7.47-7.30 (7H,m); 7.24-7.14(3H,m);7.03(1H,t); 6.27(1H,d); 5.70(1H,dd); 5.66(2H,s); 5.22(2H,s); 4.98(1H,m);4.84(1H,t); 4.82(1H,d); 4.30(1H,m); 3.90(1H,m); 2.40(2H,m); 1.58(6H,d);1.40(2H,m).

EXAMPLE 7 Synthesis of(βR,δR)-2(4-fluorophenyl)-β,¢-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbon-yl]-1H-pyrrole-1-heptanoicacid 4-(nitrooxy)butyl ester (atorvastatin 4-(nitrooxy)butyl ester)

a)(βR,δR)-2(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoicacid 4-bromobutyl ester

To a solution of 1,4-dibromobutane (0.34 ml, 2.88 mmol) inN,N-dimethylformamide (10 ml) a mixture of atorvastatin calcium (0.83 g,0.72 mmol) in N,N-dimethylformamide (10 ml) was added dropwise. Thereaction mixture was stirred at room temperature for 24 h. The solutionwas then treated with water and diethyl ether, the organic layers wereanidrified with sodium sulfate and concentrated under reduced pressure.The residue was purified by silica gel chromatography, withn-hexane/ethyl acetate 6/4 as eluent to give the title compound (0.26 g)as a white solid.

b)(βR,δR)-2(4-fluorophenyl)-β,δ-dihydroxy-5-(1-methylethyl)-3-phenyl-4-[(phenylamino)carbonyl]-1H-pyrrole-1-heptanoicacid 4-(nitrooxy)butyl ester

A solution of the compound obtained in a) above (0.25 g, 0.36 mmol) andsilver nitrate (0.38 g, 2.2 mmol) in acetonitrile (10 ml) was stirred at50° C., in the dark, for 21 h. The precipitate thus formed (silver salt)was filtered off and the solvent was evaporated under vacuum. Theresidue was then purified by silica gel chromatography, withn-hexane/ethyl acetate 1/1 as eluent to give the desired product (0.21g) was as white powder.

¹H-NMR δ (DMSO): 9.80 (1H, s); 7.51 (2H, d); 7.26-7.19 (6H, m);7.09-6.95 (6H,m); 4.73 (1H, d); 4.61 (1H, d); 4.54 (2H, t); 4.04 (2H,t); 4.00-3.70 (3H, m); 3.50 (1H, m); 3.22(1H, m); 2.40 (1H, dd); 2.25(1H, dd); 1.80-1.10 (8H, m); 1.38 (6H, d).

According to Examples 1-7 further compounds of the invention can beobtained starting from appropriate reactants and employing alsocerivastatin and rosuvastatin instead of pravastatin, fluvastatin andatorvastatin.

1. A compound of general formula (I) or a pharmaceutically acceptablesalt or stereoisomer thereof

wherein: X is —O—, —S—, —NH— or —NHR¹—, R¹ being straight or branchedalkyl with 1 to 10 carbon atoms, R is a statin residue of formula

Y is a bivalent radical having the following meaning: a) straight orbranched C₁-C₂₀ alkylene being optionally substituted with one or moreof the substituents selected from the group consisting of: halogenatoms, hydroxy, —ONO₂ or T₀, wherein T₀ is —OC(O) (C₁-C₁₀ alkyl)-ONO or—O(C₁-C₁₀ alkyl)-ONO₂; cycloalkylene with 5 to 6 carbon atoms in thecycloalkylene ring, the ring being optionally substituted with sidechains T, wherein T is straight or branched alkyl with from 1 to 10carbon atoms; b) c)

wherein n is an integer from 0 to 20, and n¹ is an integer from 1 to 20;d)

wherein: n¹ is as defined above and n² is an integer from 0 to 2;X₁═—OCO— or —COO— and R² is H or CH₃; e)

wherein: n¹, n², R² and X₁ are as defined above; Y¹ is —CH₂—CH₂— or—CH═CH— (CH₂)_(n) ²—; f)

wherein: n¹ and R² are as defined above, R³ is H or COCH₃; with theproviso that when Y is selected from bivalent radicals mentioned underb)-f), the —ONO₂ group is bound to —(CH₂)_(n) ¹; g)

wherein X₂ is —O— or —S—, n³ is an integer from 1 to 6, R₂ is as definedabove; h)

wherein: n⁴ is an integer from 0 to 10; n⁵ is an integer from 1 to 10;R⁴, R⁵, R⁶, R⁷ are the same or different, and are H or straight orbranched C₁-C₄-alkyl; wherein the —ONO₂ group is bound to

n⁵ being as defined above; Y² is a 5 or 6 member saturated, unsaturatedor aromatic heterocyclic ring, containing one or more heteroatomsselected from nitrogen, oxygen, or sulphur.
 2. A compound of generalformula (I) and/or a pharmaceutically acceptable salt or stereoisomerthereof according to claim 1 wherein: X is —O— or —S—; R is a statinresidue as defined in claim 1; Y is a bivalent radical having thefollowing meaning: a) straight or branched C₁-C₁₀ alkylene, beingoptionally substituted with T₀, wherein T₀ is as defined in claim 1; b)c)

wherein n is an integer from 0 to 5, and n¹ is an integer from 1 to 5;d)

wherein: n¹ is as defined above and n² is an integer from 0 to 2;X¹═—OCO— or —COO— and R² is H or CH₃; e)

wherein: n¹, n², R₂ and X₁ are as defined above; Y¹ is —CH═CH—; f)

wherein: n¹ and R² are as defined above, R³ is H or COCH₃; with theproviso that when Y is selected from the bivalent radicals mentionedunder b)-f), the —ONO₂ group is bound to —(CH₂)n¹; g)

wherein X₂ is —O— or —S—, n³ is an integer from 1 to 4, R² is as definedabove; h)

wherein: n⁴ is an integer from 0 to 3; n⁵ is an integer from 1 to 3; R⁴,R⁵, R⁶, R⁷ are the same and are H; and wherein the —ONO₂ group is linkedto

Y² is a 6 member saturated, unsaturated or aromatic heterocyclic ring,containing one or more atoms of nitrogen.
 3. A compound of formula (I)according to claim 1, that is pravastatin 4-(nitrooxy)butyl ester.
 4. Acompound of formula (I) according to claim 1, that is pravastatin4-(nitrooxymethyl)-benzyl ester.
 5. A compound of formula (I) accordingto claim 1, that is pravastatin 3-(nitrooxymethyl)-benzyl ester.
 6. Acompound of formula (I) according to claim 1, that pravastatin2-(nitrooxymethyl)-benzyl ester.
 7. A compound of formula (I) accordingto claim 1, that pravastatin 4-(nitrooxymethyl)-phenyl ester.
 8. Acompound of formula (I) according to claim 1, that is pravastatin3-(nitrooxymethyl)-phenyl ester.
 9. A compound of formula (I) accordingto claim 1, that is pravastatin 2-(nitrooxymethyl)-phenyl ester.
 10. Acompound of formula (I) according to claim 1, that is pravastatin2-[2′-(nitrooxy)ethyloxy]ethyl ester.
 11. A compound of formula (I)according to claim 1, that is pravastatin2-methoxy-4-[[4′-(nitrooxy)butyl]trans-2-propenoyloxy]phenol ester. 12.A method of reducing inflammatory, thrombotic and platelet activity,comprising administering to a patient in need thereof an effectiveamount of a compound according to claim
 1. 13. A method of reducingcholesterol and triglycerides levels and/or for raising high densitylipoprotein cholesterol (“HLP-C”) levels, comprising administering aneffective amount of a compound according to claim
 1. 14. A method oftreating acute coronary syndromes, stroke, peripheral vascular diseases,vascular complications in diabetic patients and atherosclerosis,comprising administering an effective amount of a compound according toclaim
 1. 15. The method according to claim 14, wherein the peripheralvascular disease is peripheral ischemia.
 16. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and apharmaceutically effective amount of a compound of general formula (I)and/or a salt or stereoisomer thereof according to claim
 1. 17. Apharmaceutical composition according to claim 16 in a suitable form forthe oral, parenteral, rectal, topic and transdermic administration, byinhalation spray or aerosol or iontophoresis devices.
 18. Liquid orsolid pharmaceutical composition for oral, parenteral, rectal, topic andtransdermic administration or inhalation in form of tablets, capsulesand pills eventually with enteric coating, powders, granules, gels,emulsions, solutions, suspensions, syrups, elixir, injectable forms,suppositories, in transdermal patches or liposomes, containing acompound of formula (I) according to claim 1 and/or a salt orstereoisomer thereof and a pharmaceutically acceptable carrier.
 19. Apharmaceutical composition comprising a compound of formula (I)according to claim 1, at least a compound used to treat cardiovasculardisease and a pharmaceutically acceptable carrier.
 20. A pharmaceuticalcomposition according to claim 19, wherein the compound used to treatcardiovascular disease is selected from the group consisting of: ACEinhibitors, angiotensin II receptor antagonists, beta-adrenergicblockers, calcium channel blockers, antithrombotics, aspirin, nitrosatedACE inhibitors, nitrosated angiotensin II receptor antagonists,nitrosated beta-adrenergic blockers and nitrosated aspirin.
 21. Apharmaceutical kit comprising the composition of claim 16 and a compoundused to treat cardiovascular disease.